Group 2i Isochrysidales thrive in marine and lacustrine systems with ice cover.

Global warming is causing rapid changes to the cryosphere. Predicting the future trajectory of the cryosphere requires quantitative reconstruction of its past variations. A recently identified sea-ice-associated haptophyte, known as Group 2i Isochrysidales, has given rise to a new sea-ice proxy with its characteristic alkenone distributions. However, apart from the occurrence of Group 2i Isochrysidales in regions with sea ice, and the empirical relationship between C37:4 alkenone abundance and sea-ice concentration, little is known about the ecology of these haptophyte species. Here, we systematically mapped the spatial and temporal occurrence of known Group 2i Isochrysidales based on environmental DNA in both marine and lacustrine environments. Our results indicate Group 2i is widely distributed in icy marine and lacustrine environments in both Northern and Southern Hemisphere, but is absent in warm environments. Temporally, Group 2i is part of the sea-ice algae bloom during the cold seasons, in contrast to other Isochrysidales that bloom in open waters during warm seasons. Our results indicate that ice is a prerequisite for the occurrence of the psychrophilic Group 2i haptophytes in marine and lacustrine ecosystems and further affirms its value for past ice reconstructions.

Cross noise level PET denoising with continuous adversarial domain generalization.

Objective.Performing positron emission tomography (PET) denoising within the image space proves effective in reducing the variance in PET images. In recent years, deep learning has demonstrated superior denoising performance, but models trained on a specific noise level typically fail to generalize well on different noise levels, due to inherent distribution shifts between inputs. The distribution shift usually results in bias in the denoised images. Our goal is to tackle such a problem using a domain generalization technique.Approach.We propose to utilize the domain generalization technique with a novel feature space continuous discriminator (CD) for adversarial training, using the fraction of events as a continuous domain label. The core idea is to enforce the extraction of noise-level invariant features. Thus minimizing the distribution divergence of latent feature representation for different continuous noise levels, and making the model general for arbitrary noise levels. We created three sets of 10%, 13%-22% (uniformly randomly selected), or 25% fractions of events from 9718F-MK6240 tau PET studies of 60 subjects. For each set, we generated 20 noise realizations. Training, validation, and testing were implemented using 1400, 120, and 420 pairs of 3D image volumes from the same or different sets. We used 3D UNet as the baseline and implemented CD to the continuous noise level training data of 13%-22% set.Main results.The proposed CD improves the denoising performance of our model trained in a 13%-22% fraction set for testing in both 10% and 25% fraction sets, measured by bias and standard deviation using full-count images as references. In addition, our CD method can improve the SSIM and PSNR consistently for Alzheimer-related regions and the whole brain.Significance.To our knowledge, this is the first attempt to alleviate the performance degradation in cross-noise level denoising from the perspective of domain generalization. Our study is also a pioneer work of continuous domain generalization to utilize continuously changing source domains.

Learn from orientation prior for radiograph super-resolution: Orientation operator transformer.

BACKGROUND AND OBJECTIVE: High-resolution radiographic images play a pivotal role in the early diagnosis and treatment of skeletal muscle-related diseases. It is promising to enhance image quality by introducing single-image super-resolution (SISR) model into the radiology image field. However, the conventional image pipeline, which can learn a mixed mapping between SR and denoising from the color space and inter-pixel patterns, poses a particular challenge for radiographic images with limited pattern features. To address this issue, this paper introduces a novel approach: Orientation Operator Transformer - O2former. METHODS: We incorporate an orientation operator in the encoder to enhance sensitivity to denoising mapping and to integrate orientation prior. Furthermore, we propose a multi-scale feature fusion strategy to amalgamate features captured by different receptive fields with the directional prior, thereby providing a more effective latent representation for the decoder. Based on these innovative components, we propose a transformer-based SISR model, i.e., O2former, specifically designed for radiographic images. RESULTS: The experimental results demonstrate that our method achieves the best or second-best performance in the objective metrics compared with the competitors at ×4 upsampling factor. For qualitative, more objective details are observed to be recovered. CONCLUSIONS: In this study, we propose a novel framework called O2former for radiological image super-resolution tasks, which improves the reconstruction model's performance by introducing an orientation operator and multi-scale feature fusion strategy. Our approach is promising to further promote the radiographic image enhancement field.

Source-free domain adaptive segmentation with class-balanced complementary self-training.

Unsupervised domain adaptation (UDA) plays a crucial role in transferring knowledge gained from a labeled source domain to effectively apply it in an unlabeled and diverse target domain. While UDA commonly involves training on data from both domains, accessing labeled data from the source domain is frequently constrained, citing concerns related to patient data privacy or intellectual property. The source-free UDA (SFUDA) can be promising to sidestep this difficulty. However, without the source domain supervision, the SFUDA methods can easily fall into the dilemma of "winner takes all", in which the majority category can dominate the deep segmentor, and the minority categories are largely ignored. In addition, the over-confident pseudo-label noise in self-training-based UDA is a long-lasting problem. To sidestep these difficulties, we propose a novel class-balanced complementary self-training (CBCOST) framework for SFUDA segmentation. Specifically, we jointly optimize the pseudo-label-based self-training with two mutually reinforced components. The first class-wise balanced pseudo-label training (CBT) explicitly exploits the fine-grained class-wise confidence to select the class-wise balanced pseudo-labeled pixels with the adaptive within-class thresholds. Second, to alleviate the pseudo-labeled noise, we propose a complementary self-training (COST) to exclude the classes that do not belong to, with a heuristic complementary label selection scheme. We evaluated our CBCOST framework on both 2D and 3D cross-modality cardiac anatomical segmentation tasks and brain tumor segmentation tasks. Our experimental results showed that our CBCOST performs better than existing SFUDA methods and yields similar performance, compared with UDA methods with the source data.

Iberian Margin surface ocean cooling led freshening during Marine Isotope Stage 6 abrupt cooling events.

The high-resolution paleoclimate records on the Iberian Margin provide an excellent archive to study the mechanism of abrupt climate events. Previous studies on the Iberian Margin proposed that the surface cooling reconstructed by the alkenone-unsaturation index coincided with surface water freshening inferred from an elevated percentage of tetra-unsaturated alkenones, C37:4%. However, recent data indicate that marine alkenone producers, coccolithophores, do not produce more C37:4 in culture as salinity decreases. Hence, the causes for high C37:4 are still unclear. Here we provide detailed alkenone measurements to trace the producers of alkenones in combination with foraminiferal Mg/Ca and oxygen isotope ratios to trace salinity variations. The results indicate that all alkenones were produced by coccolithophores and the high C37:4% reflects decrease in SST instead of freshening. Furthermore, during the millennial climate changes, a surface freshening did not always trigger a cooling, but sometimes happened in the middle of multiple-stage cooling events and likely amplified the temperature decrease.

Comprehensive analysis of alkenones by reversed-phase HPLC-MS with unprecedented selectivity, linearity and sensitivity.

Alkenones are among the most widely used paleotemperature biomarkers. Traditionally, alkenones are analyzed using gas chromatography-flame ionization detector (GC-FID), or GC-chemical ionization-mass spectrometry (GC-CI-MS). However, these methods encounter considerable challenges for samples that exhibit matrix interference or low concentrations, with GC-FID requiring tedious sample preparations and GC-CI-MS suffering from nonlinear response and a narrow linear dynamic range. Here we demonstrate that reversed-phase high pressure liquid chromatography-mass spectrometry (HPLC-MS) methods provide excellent resolution, selectivity, linearity and sensitivity for alkenones in complex matrices. We systematically compared the advantages and limitations of three mass detectors (quadrupole, Orbitrap, and quadrupole-time of flight) and two ionization modes (electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)) for alkenone analyses. We demonstrate that ESI performs better than APCI as response factors of various unsaturated alkenones are similar. Among the three mass analyzers tested, orbitrap MS provided the lowest limit of detection (0.4, 3.8 and 8.6 pg injected masses for Orbitrap, qTOF and single quadrupole MS, respectively) and the widest linear dynamic range (600, 20 and 30 folds for Orbitrap, qTOF and single quadrupole MS, respectively). Single quadrupole MS operated in ESI mode provides accurate quantification of proxy measurements over a wide range of injection masses, and with its modest instrument cost, represents an ideal method for routine applications. Analysis of global core-top sediment samples confirmed the efficacy of HPLC-MS methods for the detection and quantification of paleotemperature proxies based on alkenones and their superiority over GC-based methods. The analytical method demonstrated in this study should also allow highly sensitive analyses of diverse aliphatic ketones in complex matrices.

The first high resolution PAH record of industrialization over the past 200 years in Liaodong Bay, northeastern China.

Polycyclic aromatic hydrocarbons (PAHs) are excellent tracers for fossil fuel combustion, natural fires and petroleum contamination, and have been widely used for reconstructing past wildfires and industrial activities at a variety of time scales. Here, for the first time, we obtain a high resolution (annual to decadal scale) record of PAHs from two parallel marine sediment cores from the Liaodong Bay, Northeastern China to reconstruct the industrial activities, spanning the past ∼ 200 years from 1815 to 2014. Our data indicate that PAH variations can be divided into four episodes: I) low (probably near background) PAHs from natural fires and domestic wood combustion during the pre-industrial period from 1815 to 1890; II) slightly increased (but with large fluctuations) PAH concentrations derived from intermittent warfare during the World War (1891-1945) and increased industrial activities after 1946 (1946-1965); III) a period of stagnation and, in some cases, reduction in PAHs during the "Cultural Revolution" (1966 to 1979); and IV) a rapid and persistent rise in PAHs post 1979 linked to fast economic development, with PAH concentrations doubled from 1979 to 2014. Changes in PAH distributions demonstrate major shifts in the dominant types of fuels over time from vegetation/wood, to coal and wood, followed by coal and petroleum (including vehicle emissions) over the past 200 years. We find that PAH records also show similar trend to domestic economy and the estimated regional Anthropocene CO2 emissions from industrial activities, suggesting sedimentary PAH fluxes could be used as an indirect and qualitative proxy to track the trend for regional anthropogenic CO2 emissions.

The diagnostic value of soluble TIM-3 in oral squamous cell carcinoma.

Objective: To evaluate the soluble TIM-3 (sTIM-3) expression level in oral squamous cell carcinoma (OSCC) and determine its clinical diagnostic potential. Methods: The sTIM-3 and squamous cell carcinoma antigen (SCCAg) levels of 199 OSCC patients and 107 healthy individuals were assessed using an enzyme-linked immunosorbent assay and their individual and combined efficiency rates were compared. Results: The results showed higher sTIM-3 and SCCAg levels in the OSCC patients and better diagnostic potential for a combination of these markers than for their individual assessments, as well as positive correlation of sTIM-3 levels with clinicopathological factors. Conclusion: sTIM-3 is a potential novel and readily accessible OSCC biomarker, which in combination with SCCAg expression level might better diagnose OSCC patients.

Lay abstract Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. It affects the patients' swallowing, speech, appearance, social interactions and other aspects. Soluble TIM-3 (sTIM-3) is a negative regulatory molecule. Serum levels of squamous cell carcinoma antigen are closely related to the growth of OSCC tumors. Herein, the serum levels of sTIM-3 in OSCC patients were examined. This study demonstrated that the combined assessment of sTIM-3 and squamous cell carcinoma antigen expression levels might better distinguish OSCC patients from healthy individuals.

Elucidation of double-bond positions of polyunsaturated alkenes through gas chromatography/mass spectrometry analysis of mono-dimethyl disulfide derivatives.

RATIONALE: Derivatization with dimethyl disulfide (DMDS) followed by gas chromatography/mass spectrometry (GC/MS) analysis is a well-established method for locating double-bond position on the alkyl chain of mono-unsaturated compounds such as alkenes. For alkenes containing more than one double bond, however, the conventional DMDS derivatization approach forms poly- or cyclized DMDS adducts whose mass spectra are difficult to interpret in terms of double-bond positions. In this study, we report an efficient experimental procedure to produce mono-DMDS adducts for polyunsaturated alkenes with two to six double bonds. GC/MS analyses of these mono-DMDS adducts yield highly characteristic mass fragments, allowing unambiguous assignments of double-bond positions on the alkyl chain. We also apply our new approach (i.e., preferential formation of mono-DMDS adducts during derivatization with DMDS) to determine the double-bond positions of unsaturated alkenes produced by laboratory cultured Isochrysis litoralis, a haptophyte algal species. METHODS: Alkenes from different sources were derivatized with DMDS at 25°C for 20 to 160 min. The mass spectra of mono-DMDS adducts were obtained by GC/EI-MS analysis of reaction products which contain chromatographically resolved mono-DMDS adducts. RESULTS: Mass spectra of corresponding mono-DMDS adducts contain prominent diagnostic ions that allow a conclusive elucidation of double-bond positions. In culture samples of Isochrysis litoralis, a series of novel mono- to tri-unsaturated C31 alkenes (9-C31:1 , 6,9-C31:2 , 6,22-C31:2 , 6,25-C31:2 , 9,22-C31:2 , 6,9,25-C31:3 ) were discovered for the first time. CONCLUSIONS: A highly efficient DMDS derivatization approach is developed to yield abundant mono-DMDS adducts of polyunsaturated alkyl alkenes for elucidating double-bond positions using GC/MS.

Climate change facilitated the early colonization of the Azores Archipelago during medieval times.

Humans have made such dramatic and permanent changes to Earth's landscapes that much of it is now substantially and irreversibly altered from its preanthropogenic state. Remote islands, until recently isolated from humans, offer insights into how these landscapes evolved in response to human-induced perturbations. However, little is known about when and how remote systems were colonized because archaeological data and historical records are scarce and incomplete. Here, we use a multiproxy approach to reconstruct the initial colonization and subsequent environmental impacts on the Azores Archipelago. Our reconstructions provide unambiguous evidence for widespread human disturbance of this archipelago starting between 700-60+50 and 850-60+60 Common Era (CE), ca. 700 y earlier than historical records suggest the onset of Portuguese settlement of the islands. Settlement proceeded in three phases, during which human pressure on the terrestrial and aquatic ecosystems grew steadily (i.e., through livestock introductions, logging, and fire), resulting in irreversible changes. Our climate models suggest that the initial colonization at the end of the early Middle Ages (500 to 900 CE) occurred in conjunction with anomalous northeasterly winds and warmer Northern Hemisphere temperatures. These climate conditions likely inhibited exploration from southern Europe and facilitated human settlers from the northeast Atlantic. These results are consistent with recent archaeological and genetic data suggesting that the Norse were most likely the earliest settlers on the islands.

Study on energy saving and environmental protection generator with hydrogen-oxygen-gasoline compound fuel.

The generator is the most popular mobile power device and backup power device in the world. It is very important for human life. Therefore, it is important to develop more efficient combustion technology in order to save energy and reduce air pollution. In this paper, a novel technology of hydrogen and oxygen compound gasoline fuel is developed. Hydrogen and oxygen gases are produced from an electrolytic cell and then mixed with the intake gasoline and air. The compound fuel is sucked into the engine combustion chamber. The hydrogen and oxygen gases can be produced immediately without any storage device of hydrogen. The experimental results show that this technology can increase the power generation and decrease emission pollution due to promoting combustion efficiency. In addition, the spark plug seat temperature increases due to higher heat value of hydrogen. This technique can reduce carbon monoxide and HC, but increase carbon dioxide. The research and development of this technique can achieve the goals of energy saving, emission reduction, relative safety, easy refitting and low refitting expense. Moreover, this research possesses academic innovation and industrial application.

Inverse hydrogen isotope fractionation indicates heterotrophic microbial production of long-chain n-alkyl lipids in desolate Antarctic ponds.

Long-chain (>C25 ) n-alkyl lipids have long been considered biomarkers for higher plant leaf waxes and widely applied for paleoclimate and paleoenvironmental reconstructions. However, recent experimental and lacustrine sediment studies suggest long-chain n-alkanes and n-alkanoic acids can also be produced by aerobic microbes, probably heterotrophic microbes based on carbon isotope data. Here we show that sedimentary long-chain n-alkanes and n-alkanoic acids in two desolate Antarctic ponds where vascular plants are absent in the surroundings display hydrogen isotopic values up to 300 per mil higher than those of lake water. It is the first time that such strongly inverse or reduced hydrogen isotopic fractionation of lipid biomarkers is observed in natural sediment samples. Based on recent extensive experimental data on microbial hydrogen isotopic fractionation, our data can only be explained by the predominant production of long-chain n-alkanes and n-alkanoic acids from heterotrophic micro-organisms. Together with preliminary 16S rRNA gene sequencing data, our results represent the first unambiguous example of predominant heterotrophic microbial production of long-chain n-alkyl waxes in a natural environment.

Silver (I)-dimercaptotriazine functionalized silica: A highly selective liquid chromatography stationary phase targeting unsaturated molecules.

Silver(I)-mercaptopropyl (Ag-MP) functionalized silica gel has demonstrated its effectiveness in separating various unsaturated organic compounds including unsaturated fatty acid ethyl esters (FAEEs), triglycerols (TAGs) and long-chain alkyl ketones (alkenones). While Ag-MP stationary phase displays many advantages over the conventional silver ion-impregnated silica gel (e.g., stability, high recovery, etc.), potential drawbacks of Ag-MP include relatively low retentions for unsaturated molecules, which could limit chromatographic resolutions under certain circumstances. In this study, we evaluate a new silver-thiolate stationary phase: silver(I)-dimercaptotriazine (Ag-DMT) functionalized silica gel targeting the separation of unsaturated compounds. We show Ag-DMT affords substantially higher retention factors, peak resolutions and capacities for TAGs and FAEEs than Ag-MP does. Ag-DMT also yields higher purity eicosapentaenoic acid (EPA) from fish oil FAEE mixtures than Ag-MP. In addition, Ag-DMT resolves double bond positional and cis/trans-isomers of C18:1 fatty acid methyl esters (FAMEs) as well as unsaturated methyl/ethyl alkenones with different number of double bonds. Based on van't Hoff plots, enthalpy changes during the adsorption of unsaturated FAEEs onto Ag-DMT are ~2 times higher than those on Ag-MP. Such difference may be attributed to the stronger electron-withdrawing effect of the thiol group on DMT, which results in more positively charged silver ions hence greater interactions with unsaturated molecules. The stronger interaction between double bonds and Ag-DMT is further corroborated by density-functional theory (DFT) calculations. Ag-DMT shows its high stability for repeated uses in the separation of TAGs over 319 runs, with peak resolutions decreasing by < 3%. Collectively, our data demonstrate the exceptionally high efficiency of Ag-DMT column for separating unsaturated molecules.

Group 2i Isochrysidales produce characteristic alkenones reflecting sea ice distribution.

Alkenones are biomarkers produced solely by algae in the order Isochrysidales that have been used to reconstruct sea surface temperature (SST) since the 1980s. However, alkenone-based SST reconstructions in the northern high latitude oceans show significant bias towards warmer temperatures in core-tops, diverge from other SST proxies in down core records, and are often accompanied by anomalously high relative abundance of the C37 tetra-unsaturated methyl alkenone (%C37:4). Elevated %C37:4 is widely interpreted as an indicator of low sea surface salinity from polar water masses, but its biological source has thus far remained elusive. Here we identify a lineage of Isochrysidales that is responsible for elevated C37:4 methyl alkenone in the northern high latitude oceans through next-generation sequencing and lab-culture experiments. This Isochrysidales lineage co-occurs widely with sea ice in marine environments and is distinct from other known marine alkenone-producers, namely Emiliania huxleyi and Gephyrocapsa oceanica. More importantly, the %C37:4 in seawater filtered particulate organic matter and surface sediments is significantly correlated with annual mean sea ice concentrations. In sediment cores from the Svalbard region, the %C37:4 concentration aligns with the Greenland temperature record and other qualitative regional sea ice records spanning the past 14 kyrs, reflecting sea ice concentrations quantitatively. Our findings imply that %C37:4 is a powerful proxy for reconstructing sea ice conditions in the high latitude oceans on thousand- and, potentially, on million-year timescales.

The impact of anthropogenic activity at the tropical Sergipe-Poxim estuarine system, Northeast Brazil: Fecal indicators.

The individual concentrations and sources of sterols in sediments samples collected in two periods 2017 (dry period /March and rainy period/August) were determined along with the Sergipe-Poxim estuarine system, Aracaju, Brazil. The individual sterols concentration ranged from 135 to 21,746 ng g-1 (March) and 191 to 144,748 ng g-1 (August) and the distribution was mainly dominated by ß-sitosterol in both periods with 37.2% (March) and 70.8% (August) of the total sterols found. In all the sampling sites, the coprostanol levels were higher than 100 ng g1 (March) and 500 ng g-1 (August), indicating sewage contamination. Diagnostic ratios between sterols suggested the predominance of sewage sources. Pearson correlation assessed a correlation significant (March) and negligible (August) between coprostanol concentration levels and organic matter. The principal component analysis (PCA) showed that the sterols levels influenced strongly C1, as well as C2 distinguished between the plant sterols and from sewage.

An efficient approach to eliminate steryl ethers and miscellaneous esters/ketones for gas chromatographic analysis of alkenones and alkenoates.

Long-chain alkenones (LCAs) and alkenoates (LCEs) are highly valuable biomarkers for paleotemperature reconstructions. A major problem, however, for accurate quantification of these compounds using gas chromatography (GC) is co-elution with steryl ethers, wax esters, saturated ketones and other numerous mid-polarity compounds frequently encountered in marginal marine and lake sediments. Co-elution during GC separation is prevalent, particularly if the full homologous series of alkenones and alkenoates are to be analyzed. Taking advantage of the presence of two or more double bonds in LCAs and LCEs, the conventional silica gel impregnated with silver nitrate has previously been used to remove co-eluting compounds for LCAs. However, this conventional argentation chromatography is hampered by the extreme instability of silver nitrate, poor reproducibility, low recovery and short lifetime. Here we demonstrate a highly efficient flash chromatographic approach based on silver thiolate chromatographic material (AgTCM) that overcomes the shortcomings of the traditional argentation chromatography and allows repeated sample preparation (up to 62 samples in one test) with little loss in separation efficiency. AgTCM selectively extracts LCAs and LCEs and effectively eliminates co-eluting compounds including steryl ethers and wax esters for the subsequent gas chromatography (GC) analysis. This new method, therefore, allows low-cost and high-throughput sample preparation for comprehensive quantification of the full homologous series of LCAs and LCEs in marine and lake sediments.

Phylogenetic diversity in freshwater-dwelling Isochrysidales haptophytes with implications for alkenone production.

Members of the order Isochrysidales are unique among haptophyte lineages in being the exclusive producers of alkenones, long-chain ketones that are commonly used for paleotemperature reconstructions. Alkenone-producing haptophytes are divided into three major groups based largely on molecular ecological data: Group I is found in freshwater lakes, Group II commonly occurs in brackish and coastal marine environments, and Group III consists of open ocean species. Each group has distinct alkenone distributions; however, only Groups II and III Isochrysidales currently have cultured representatives. The uncultured Group I Isochrysidales are distinguished geochemically by the presence of tri-unsaturated alkenone isomers (C37:3b Me, C38:3b Et, C38:3b Me, C39:3b Et) present in water column and sediment samples, yet their genetic diversity, morphology, and environmental controls are largely unknown. Using small-subunit (SSU) ribosomal RNA (rRNA) marker gene amplicon high-throughput sequencing of environmental water column and sediment samples, we show that Group I is monophyletic with high phylogenetic diversity and contains a well-supported clade separating the previously described "EV" clade from the "Greenland" clade. We infer the first partial large-subunit (LSU) rRNA gene Group I sequence phylogeny, which uncovered additional well-supported clades embedded within Group I. Relative to Group II, Group I revealed higher levels of genetic diversity despite conservation of alkenone signatures and a closer evolutionary relationship with Group III. In Group I, the presence of the tri-unsaturated alkenone isomers appears to be conserved, which is not the case for Group II. This suggests differing environmental influences on Group I and II and perhaps uncovers evolutionary constraints on alkenone biosynthesis.

Discovery of alkenones with variable methylene-interrupted double bonds: implications for the biosynthetic pathway.

Alkenones (C37 -C40 ) are highly specific biomarkers produced by certain haptophyte algae in ocean and lacustrine environments and have been widely used for paleoclimate studies. Unusual shorter-chain alkenones (SCA; e.g., C35 and C36 ) have been found in environmental and culture samples, but the origin and structure of these compounds are much less understood. The marine alkenone producer, Emiliania huxleyi CCMP2758 strain, was reported with abundant C35:2 Me (∆12, 19 ) alkenones when cultured at 15°C (Prahl et al. 2006). Here we show, when this strain is cultured at 4°C-10°C, that CCMP2758 produces abundant C35:3 Me, C36:3 Me, and small amounts of C36:3 Et alkenones with unusual double-bond positions of ∆7, 12, 19 . We determine the double-bond positions of the C35:3 Me and C36:3 Me alkenones by GC-MS analysis of the dimethyl disulfide and cyclobutylamine derivatives, and we provide the first temperature calibrations based on the unsaturation ratios of the C35 and C36 alkenones. Previous studies have found C35:2 Me (∆14, 19 ) and C36:2 Et (∆14, 19 ) alkenones with three-methylene interruption in the Black Sea sediments, but this is the first reported instance of alkenones with a mixed three- and five-methylene interruption configuration in the double-bond positions. The discovery of these alkenones allows us to propose a novel biosynthetic scheme, termed the SCA biosynthesis pathway, that simultaneously rationalizes the formation of both the C35:3 Me (∆7, 12, 19 ) alkenone in our culture and the ∆14, 19 Black Sea type alkenones without invoking new desaturases for the unusual double-bond positions.

Identification of double-bond positions in isomeric alkenones from a lacustrine haptophyte.

RATIONALE: Measurements of alkenone unsaturation ratios are widely used for paleotemperature reconstructions in ocean and lake environments. Previously, we reported the discovery of a series of tri-unsaturated alkenone positional isomers (Δ(14, 21, 28) ) from oligosaline and freshwater lakes in Greenland and Alaska. In this work we provide a detailed analysis of the structures and isotopic compositions (δ(13) C and δ(2) H) of the alkenones produced by the "Greenland haptophyte". METHODS: Alkenones were extracted from sediments of Lake BrayaSø, Greenland. Alkenone double-bond positions were determined by GC/EI-MS analysis of alkenone dimethyl disulfide and cyclobutylimine derivatives. Alkenones were purified by semi-preparative HPLC using a silver(I) thiolate stationary phase. Carbon and hydrogen isotope analysis was performed by gas chromatography/isotope ratio mass spectrometry (GC/IRMS). RESULTS: A series of novel tri-unsaturated alkenone positional isomers were identified among four alkenone homologues (i.e. C37 Me , C38 Me , C38 Et , and C39 Et ) with double-bond positions at Δ(14, 21, 28) . The hydrogen isotope compositions (δ(2) H, VSMOW) of the tri-unsaturated positional isomers from C37 Me and C38 Et were slightly depleted (~ -11 ‰) relative to the common tri-unsaturated alkenone. The carbon isotope composition (δ(13) C, VPDB) of the tri-unsaturated positional isomers from the C37 Me , C38 Me , C38 Et , and C39 Et alkenones were significantly enriched (~ +4 ‰) relative to the common alkenones (di-, tri-, and tetra-unsaturated). CONCLUSIONS: The novel tri-unsaturated alkenone positional isomers produced by the Greenland haptophyte possess Δ(14, 21, 28) double-bond positions, instead of the common Δ(7, 14, 21) double-bond positions. The hydrogen isotope values suggest the novel tri-unsaturated positional isomers could be biosynthetic precursors to the tetra-unsaturated alkenones (Δ(7, 14, 21, 28) ). However, the significantly higher carbon isotope values of the tri-unsaturated positional isomers relative to the common di-, tri- and tetra-unsaturated alkenones suggest these positional isomers may have different/additional biosynthetic precursors.